Bae Dongsoon, Camilli Tura C, Ha Ngoc-Tram, Ceryak Susan
Department of Pharmacology and Physiology, The George Washington University Medical Center, Washington, DC 20037, United States.
Cell Signal. 2009 May;21(5):727-36. doi: 10.1016/j.cellsig.2009.01.011. Epub 2009 Jan 8.
Our recent studies showed that maintenance of protein tyrosine phosphorylation by PTP inhibition enhanced cell growth, clonogenic survival, and mutagenesis after a single low-level Cr(VI) exposure, thereby suggesting that tyrosine phosphorylation-dependent signaling may govern inappropriate survival in human lung fibroblasts (HLFs). Our goal is to identify specific phospho-tyrosine regulator(s)/ downstream effectors involved in enhanced survival after Cr(VI) exposure and PTP inhibition. Phosphotyrosine profiling array showed that PTP inhibition following Cr(VI) exposure increased tyrosine phosphorylation of specific proteins, such as FGR and ABL, which are upstream regulators of both Erk and Akt pathways. To explore the roles of these pathways in the PTP-induced increase in clonogenic survival after Cr(VI) exposure, we examined the effect of combined Akt1 and Erk1/2 knockdown via siRNA technology. Akt1 and/or Erk1/2 silencing had no effect on the PTP inhibitor-induced increase in survival following Cr(VI) exposure, suggesting the presence of non-Akt/non-Erk-mediated survival signaling. Interestingly, geldanamycin, an HSP90 inhibitor and non-specific Raf inhibitor, abrogated the PTP inhibitor-mediated increase in survival following Cr(VI) exposure and abolished the expression/activity of c-Raf and activity of Mek. These findings prompted us to explore upstream regulators of Erk, i.e., Ras, c-Raf and Mek for their potential roles in clonogenic survival. GW5074, a specific c-Raf kinase inhibitor did not alter the effect of the PTP inhibitor but decreased Cr(VI)-mediated clonogenic lethality, potentially though Mek hyperactivation. A genetic approach with a c/a Mek1 mutant also showed that Mek activity was not directly associated with the PTP inhibitor effect. Finally, a genetic approach with d/n or c/a Ras and c-Raf mutants, showed that Ras and c-Raf activities play a substantive role in enhancing clonogenic survival by PTP inhibition following Cr(VI) insult. In conclusion, these studies highlight a novel pro-survival mechanism for clonogenic survival in the face of genotoxic stress in the presence of PTP inhibition via an Erk/Mek-independent and Ras/c-Raf-dependent regulation in normal human lung fibroblasts.
我们最近的研究表明，通过抑制蛋白酪氨酸磷酸酶（PTP）来维持蛋白酪氨酸磷酸化，可增强细胞在单次低剂量六价铬（Cr(VI)）暴露后的生长、克隆存活及诱变能力，从而表明酪氨酸磷酸化依赖性信号传导可能在人肺成纤维细胞（HLFs）的异常存活中起主导作用。我们的目标是确定在Cr(VI)暴露和PTP抑制后参与增强存活的特定磷酸化酪氨酸调节因子/下游效应器。磷酸酪氨酸谱分析阵列显示，Cr(VI)暴露后抑制PTP会增加特定蛋白（如FGR和ABL）的酪氨酸磷酸化，这些蛋白是Erk和Akt通路的上游调节因子。为了探究这些通路在Cr(VI)暴露后PTP诱导的克隆存活增加中的作用，我们通过小干扰RNA（siRNA）技术检测了联合敲低Akt1和Erk1/2的效果。敲低Akt1和/或Erk1/2对PTP抑制剂诱导的Cr(VI)暴露后存活增加没有影响，这表明存在非Akt/非Erk介导的存活信号。有趣的是，格尔德霉素（一种HSP90抑制剂和非特异性Raf抑制剂）消除了PTP抑制剂介导的Cr(VI)暴露后存活增加，并消除了c-Raf的表达/活性以及Mek的活性。这些发现促使我们探究Erk的上游调节因子，即Ras、c-Raf和Mek在克隆存活中的潜在作用。GW5074（一种特异性c-Raf激酶抑制剂）并未改变PTP抑制剂的作用，但降低了Cr(VI)介导的克隆致死率，可能是通过Mek的过度激活。使用c/a Mek1突变体的遗传学方法也表明，Mek活性与PTP抑制剂的作用没有直接关联。最后，使用d/n或c/a Ras和c-Raf突变体的遗传学方法表明，在Cr(VI)损伤后，通过抑制PTP增强克隆存活的过程中，Ras和c-Raf的活性发挥了重要作用。总之，这些研究突出了一种新的促存活机制，即在正常人类肺成纤维细胞中，在PTP抑制存在的情况下，通过不依赖Erk/Mek和依赖Ras/c-Raf的调节，在基因毒性应激下实现克隆存活。
